: Glia perform a variety of important roles in controlling and supporting neurotransmission in the nervous system. The Drosophila midline glia play a unique role in CNS development by controlling the guidance of commissural axons that cross the midline. These glia are derived from bipotential glial/neuronal precursors that migrate in a characteristic manner to form the glial scaffold that ensheaths commissural axons. Over 50 genes have been identified that are expressed in midline glia. Thus, the midline glia represent an excellent system to comprehensively study the regulatory pathways that control glial development and function. The Drosophila midline glia share functional characteristics with the vertebrate floorplate cells that reside at the midline of the spinal cord. The floorplate cells also play important roles in controlling the development of specific neuronal cell types, directing axon migration, and forming a scaffold surrounding the commissural axons. Consequently, our studies have relevance to issues of human health due to the importance of glia and floorplate cells for proper nervous system development and function. The major goals of this proposal are to understand the functions of Drosophila midline glia, and how they migrate and interact with neurons and axons to form the mature glial scaffolding. Mechanistic cellular insight into midline glial migration will be determined using advanced imaging techniques we have developed, including live imaging to visualize midline glia in vivo. Midline migration and ensheathment are mediated by two cell adhesion proteins, Wrapper and Neurexin IV. Genetic experiments will screen for additional genes involved in adhesion, signaling, and other processes that regulate and mediate midline glial development. Another goal is to understand the transcriptional circuitry that controls midline glial development. The use of ChIP-Seq will identify the transcriptional target sites of Single-minded throughout the genome, thus revealing how this master regulatory protein controls transcription and development. The interactions between Single-minded and Suppressor of Hairless (another important regulator of midline glial development and the downstream effector of Notch signaling) will be analyzed using genetic and molecular techniques. Transgenic approaches will be employed to identify and analyze midline glial cis-regulatory modules to mechanistically explore how midline glial transcription is controlled. Together, this project will provide a comprehensive view regarding how transcriptional circuitry, signaling pathways, and adhesion proteins control the complex morphogenetic processes required for midline glia to interact with axons and form a glial scaffolding.